Zhao BMC Cell Biology 2016, 17(Suppl 1):16 DOI 10.1186/s12860-016-0095-7

REVIEW Open Access Expression and function of pannexins in the inner ear and hearing Hong-Bo Zhao

From International Conference 2015 Valparaiso, Chile. 28 March - 2 April 2015

Abstract Pannexin (Panx) is a family encoding gap junction in vertebrates. So far, three isoforms (Panx1, 2 and 3) have been identified. All of three Panx isoforms express in the cochlea with distinct expression patterns. Panx1 expresses in the cochlea extensively, including the spiral limbus, the organ of Corti, and the cochlear lateral wall, whereas Panx2 and Panx3 restrict to the basal cells of the stria vascularis in the lateral wall and the cochlear bony structure, respectively. However, there is no pannexin expression in auditory sensory hair cells. Recent studies demonstrated that like connexin gap junction gene, Panx1 deficiency causes hearing loss. Panx1 channels dominate ATP release in the cochlea. Deletion of Panx1 abolishes ATP release in the cochlea and reduces endocochlear potential (EP), auditory receptor current/potential, and active cochlear amplification. Panx1 deficiency in the cochlea also activates caspase-3 cell apoptotic pathway leading to cell degeneration. These new findings suggest that pannexins have a critical role in the cochlea in regard to hearing. However, detailed information about pannexin function in the cochlea and Panx mutation induced hearing loss still remain largely undetermined. Further studies are required. Keywords: Pannexin, ATP release, Endocochlear potential, Active cochlear amplification, Cell degeneration, Hearing loss, Inner ear

Background Panx1 gene is located on human Gap junction is an intercellular channel and exists in 11q14.3 in a 700 kb interval between the both vertebrates and invertebrates. However, the gap CRSP6 and MRE11, Panx2 on human chromosome junctional proteins in vertebrates and invertebrates are 22q13.31–q13.33, and Panx3 on human chromosome encoded by different genes. In vertebrates, gap junctional 11q24.2 in a 150 kb interval between the telomeric proteins are encoded by a connexin gene family, whereas border of the cluster of olfactory gene family 8 and in invertebrates they are encoded by an unrelated TBRG1. In the mouse, Panx1, 2, and 3 genes are lo- innexin gene family. About 15 years ago, by application cated on chromosome 9, 15, and 9, respectively [3]. of genoinformatics, an innexin homologue, termed pan- Phylogenetic analysis demonstrates that pannexin is nexin, was found in the [1–3]. Later, highly conserved in Nematoda, Mollusca, Arthropoda Panx expression in rodents, zebrafish, and an inverte- and mammals [1, 3], implying that pannexins may brate chordate was also identified [3]. have important functions.

Pannexin genomics and expression Pannexin channels and functions So far, three pannexin isoforms (Panx1, 2, and 3) have Despite the lack of similar sequences with connexins, been cloned from the human and mouse genomes [2, 3]. pannexin proteins share large similarities at the struc- tural and functional levels. Pannexin proteins also pos- Correspondence: [email protected] Department of Otolaryngology, University of Kentucky Medical Center, 800 sess four transmembrane domains, two extracellular Rose Street, Lexington, KY 40536, USA loops, one intracellular loop, and intracellular N- and

© 2016 Zhao. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhao BMC Cell Biology 2016, 17(Suppl 1):16 Page 122 of 150

C-termini [3]. The profile of pannexin channel per- the hearing loss appeared progressive, moderate to se- meability is similar to that of connexin channels, per- vere, and severe at high-frequency range (Fig. 2, also see meable to ions and small molecules up to 1 kDa [4]. [20, 21]). Although pannexin mutation induced hearing However, despite general parallels with connexin loss has not been identified yet, our findings strongly channels, the properties and pharmacology of pan- suggest that pannexin mutations also can induce hearing nexin channels are distinct [5, 6]. First, it is becoming loss in humans. apparent that unlike connexins to form integrated gap junction channels, pannexins mainly function as plasma Pannexin function in the cochlea membrane channels ('hemichannels') on the cell surface Panx1 channels dominate ATP release in the cochlea to provide an intracellular-extracellular conduit [7–10]. A major function of Panx1 channels is to release ATP Second, Panx1 channels demonstrate larger currents [17]. ATP is an important energy source in cells and also with increasing depolarization, faster kinetics of pore acts as an important cell signaling molecule in the extra- opening, larger unitary conductance (~500 pS, com- cellular space, when it is released to the outside of cells. pared to a maximum ~ 300 pS in Cx43 hemichannels), As mentioned above, pannexin channels possess rela- very weak voltage gating, and multiple substates in tively large pore size and are permeable to ATP. Due to single-channel recordings [2, 11, 12]. Third, both Panx1 channels can work at normal physiological levels homomeric and heteromeric (Panx1/Panx2) channels of Ca++, Panx1 channels in many organs and tissues act show significantly higher sensitivity to carbenoxolone as a major conduit for ATP release under physiological and probenecid [13, 14]. Finally, in contrast to the con- conditions [22–25]. nexin channels, which are highly sensitive to Ca++ and In the cochlea, ATP physiologically exists in the can be closed by the physiological levels of extracellular endolymph and perilymph [26]. We previously found Ca++ (1–2 mM), pannexin channels are insensitive to that gap junctional hemichannels are responsible for Ca++ and can open and function at the physiological ATP release in the cochlea [27]. However, it was extracellular levels of Ca++ [11]. These specific proper- unclear at that time which hemichannels were re- ties of the channel activity imply that pannexin chan- sponsible for ATP release. Recently, we found that nels can play an important role in a wider range of deletion of Panx1 abolished ATP release in the coch- physiological function and pathological processes. So lea, whereas deletion of Cx26 and Cx30, which are far, pannexins have been found to play important func- predominant connexin isoforms in the cochlea [28, tions in the ATP release, Ca++ wave propagation, vaso- 29], had little effect on ATP release under physio- dilation, ischemic cell death, inflammatory response, logical conditions (Fig. 3, also see [21]). Moreover, it and release of synaptic neurotransmitters [15–17]. This has been found that a gap junction channel antagon- review mainly focuses on the expression and function ist carbenoxolone (CBX) could eliminate ATP release of pannexins in the inner ear and in hearing. in the cochlea at the physiological level of extracellu- lar Ca++ (2 mM) (Fig. 3, also see [21]). These new Pannexin expression in the cochlea data demonstrate that ATP in the cochlea is mainly Like connexins, pannexins have ubiquitous expression. released via Panx1 channels under physiological In the mammalian cochlea, we found that all three conditions. pannexin isoforms have expressions [18]. Panx1 ex- These new findings, however, are inconsistent with a presses at the cochlear supporting cells, the spiral previous report that Paxn1 “knockout” had no effect on limbus, and the cochlear lateral wall. Panx2 only ex- ATP release in the cochlea [30]. In that study, ATP re- presses at the basal cell layer in the stria vascularis in lease was recorded by use of ATP sensory electrodes, the cochlear lateral wall, and Panx3 expression is re- which sensitivity is only at the micromolar levels and is stricted to the cochlear bony structure (Fig. 1). How- too low to measure fmole to submicromolar levels of ever, like connexins, the auditory sensory hair cells ATP in the cochlea [21, 26, 27]. Moreover, their used have no pannexin expression (Fig. 1, also see [18]). “Panx1 KO” mice were created by Cre-loxP technique. These distinct expression patterns strongly suggest There is no information available for which Cre mouse that pannexins have important functions in the inner line was used. Also, there were no experiments to check ear and in hearing. Panx1 expression in the cochlea. So, whether Panx1 was really deleted or not in the cochlea in that Panx1 ‘KO’ Pannexin deficiency induced hearing loss mouse remains unclear. In our experiments [21], we It has been well-known that connexin mutations can used a bioluminescent-based method to measure ATP cause hearing loss [19]. Our recent studies showed that release, which is the most sensitive and reliable method Panx1 deficiency also induces hearing loss [20, 21]. We for measuring ATP release. We also used immunofluor- found that the Panx1 deficient mice have hearing loss; escent staining and confirmed that Panx1 was deleted in Zhao BMC Cell Biology 2016, 17(Suppl 1):16 Page 123 of 150

Fig. 1 Immunofluorescent labeling for Panx1, 2, and 3 in the cochlea. a-b: Immunofluorescent staining for Panx1. Outer hair cells (OHCs) are visualized by prestin staining (red) in (panel b). c-d: Immunofluorescent staining for Panx2. e-f: Immunofluorescent staining for Panx3. HC: Hensen cell; MO: modiolus; OC: organ of Corti; RM: Reissner’s membrane; SG: spiral ganglion; SLM: spiral limbus; SP: spiral prominence; SPL: spiral ligament; SV: stria vascularis. Scale bar: 50 μmin(a, c), 100 μminE,10μmin(b, d and f). Modified from [18] the cochlea [20, 21]. Finally, we found that Cx26 and potential, thereby initiating hearing. Positive EP is Cx30 deletion had little effect on ATP release in the generated in the cochlear lateral wall. ATP is required cochlea under physiological conditions (Fig. 3, also see for EP generation [31]. We found that deletion of [21]). Thus, these new experiments and data provide Panx1 in the cochlear lateral wall reduced ATP re- strong evidence that Panx1 channels in the cochlea are re- lease and EP generation, thereby reducing auditory sponsible for ATP release under physiological conditions. receptor potential and causing hearing loss [21]. However, the detailed mechanisms for Panx1 channel ATP release and EP generation still remain unclear Ensuring of endocochlear potential and auditory receptor and require further studies in future. current/potential generation in the cochlea Positive endocochlear potential (EP, +100 ~ 110 mV) in the cochlea is indispensable for hearing and is a The role of Panx1 in active cochlear amplification driving force that compels K+ ions in the endolymph We also found that deletion of Panx1 expression in the through the transduction channels at stereocilia of cochlea could reduce active cochlear amplification [20]. hair cells to produce auditory receptor current and Distortion product otoacoustic emission (DPOAE) was

Fig. 2 Panx1 deletion induced hearing loss. a: Hearing loss as measured by ABR thresholds, which are significantly increased in Panx1 KO mice. The increase is large at high-frequency range. b: Hearing loss is progressive. **P < 0.001, two-way ANOVA with a Bonferroni correction. Modified from [20, 21] Zhao BMC Cell Biology 2016, 17(Suppl 1):16 Page 124 of 150

Fig. 3 Panx1 channels dominate ATP release in the cochlea. Deletion of Panx1 and application of 0.1 mM carbenoxolone (CBX) but not deletion of Cx26 and Cx30, which are predominant connexin isoforms in the cochlea, eliminate ATP release in the cochlea. ATP release was measured in the normal extracellular solution which contains 2 mM Ca++.**P < 0.001, one-way ANOVA with a Bonferroni correction. Modified from [21] reduced. Consistent with hearing loss, the reduction was se- deficiency leading to ATP release reduction may have vere at high frequencies [20, 21]. DPOAE in mammals is more broad effects on the cochlear function and mainly produced by outer hair cell electromotility based ac- hearing. tive cochlear amplification. However, as mentioned above (Fig. 1b, also see [18]), outer hair cells have no any pan- The role of Panx1 in cell degeneration in the cochlea nexin expression. Currently, the detailed mechanisms for One important function of pannexin channels is to how Panx1 deficiency can affect outer hair cell electromoti- participate in cell apoptotic process. It has been re- lity and reduce active cochlear amplification remain un- ported that the activation of Caspase-3 cell apoptotic clear. It could be a consequence from EP and auditory pathway can permanently open Panx1 channels lead- receptor current/potential reduction due to Panx1 knock- ing to cell apoptosis and death [46–50]. Recently, we out reduced ATP release (Fig. 3 and Fig. 7 in [21]). found that Panx1 deletion also activates Caspase-3 apoptotic pathway in the cochlea leading to cell de- Other downstream effects of ATP release reduction due to generation [20]. The activity of caspase-3 was detect- Panx1 deficiency in the cochlea able in both hair cells and cochlear supporting cells Extracellular ATP is an important extracellular cell sig- in Panx1 knockout (KO) mice. However, hair cells naling molecule and can activate purinergic P2 receptors have neither connexin nor pannexin expression [18, to play broad roles in many physiological functions and 29]. How Panx1 deletion causes hair cell degeneration pathological processes [32, 33]. In the cochlea, ATP can currently remains unknown. elevate intracellular Ca++ concentration in hair cells modifying sound transduction and neurotransmission Conclusions [34], mediate hearing sensitivity and extent the dynamic Like connexins, pannexins also have extensive expres- range of hearing [35–37], and synchronize auditory sion in the cochlea [18]. Panx1 is a predominant iso- nerve activity during development [38, 39]. In addition, form. Our recent studies showed that Panx1 deficiency ATP can also activate purinergic P2X receptors to medi- causes hearing loss, abolishes ATP release in the coch- ate stimulation of parasensory cation absorption in the lea, and reduces EP and auditory receptor potential [20, cochlea [40]. We also found that ATP can activate P2X 21]. Panx1 deficiency also activates caspase-3 cell apop- receptors to mediate outer hair cell electromotility [27, totic pathway in the cochlea leading to cell degeneration 41], gap junctional coupling [42], K+-sinking [43], and [20]. However, pannexin function in the cochlea and in EP generation [21]. Recently, it has been found that mu- hearing still remains largely undetermined. Connexin tations of P2X2 purinergic receptors induce autosomal mutations can induce a high incidence of hearing loss, dominant nonsyndromic hearing loss DFNA41 [44, 45] responsible for >50% of nonsyndromic deafness [19]. and increase susceptibility to noise stress [44], indicating These new findings strongly suggest that Panx1 muta- that Panx1-ATP-P2X receptor-mediated purinergic cell tions may also be able to induce hearing loss in humans, signaling has a critical role in hearing. Thus, Panx1 which requires further study in future. Zhao BMC Cell Biology 2016, 17(Suppl 1):16 Page 125 of 150

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